Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.     

Membrane interactions of antimicrobial peptides from Australian frogs

The membrane interactions of four antimicrobial peptides, aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, isolated from Australian tree frogs, are reviewed. All four peptides are amphipathic α-helices with a net positive charge and range in length from 13 to 25 residues. Despite several similar sequence characteristics, these peptides compromise the integrity of model membrane bilayers via different mechanisms; the shorter peptides exhibit a surface interaction mechanism while the longer peptides may form pores in membranes.     

三条新颖短肽筛选及抑菌作用的初报

[目的]为了发现新的农作物病原菌抗菌肽,人工设计并构建了大容量短肽文库,从中筛选并合成96条短肽用于鉴定其对农作物病原菌的抑菌活性.[方法]采用琼脂扩散法,对靶标菌一棉花枯萎病菌(Fusarium f.sp.vasinfecum)、棉花红腐病菌(Fusarium moniliforme)、小麦根腐病菌(Bipolaris sorokiniana)和马铃薯早疫病菌(Alternaria solani)进行抑菌初筛,并测定了有抗菌作用短肽的最小抑菌浓度和抑菌持久性.[结果]得到了A6、D4和F10对上述四种病原真菌抑菌效果较强,抑菌时间较长的抗菌肽,通过与抗菌肽数据库氨基酸序列对比,未见这3条抗菌肽的同源序列.[结论]研制的3条短肽属于新颖抗菌肽,为防治农作物真菌病害提供了新的基因资源.     

Membrane interactions of antimicrobial peptides from Australian tree frogs

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic α-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.     

Membrane interactions of antimicrobial peptides from Australian tree frogs

The skin secretions of amphibians are rich in host defence peptides. The membrane interactions of the antimicrobial peptides, aurein 1.2, citropin 1.1 and maculatin 1.1, isolated from Australian tree frogs, are reviewed. Although all three peptides are amphipathic alpha-helices, the mode of action of these membrane-active peptides is not defined. The peptides have a net positive charge and range in length from 13 to 21 residues, with the longest, maculatin 1.1, having a proline at position 15. Interestingly, alanine substitution at Pro-15 leads to loss of activity. The effects of these peptides on phospholipid bilayers indicate different mechanisms for pore formation and lysis of model membranes, with the shorter peptides exhibiting a carpet-like mechanism and the longest peptide forming pores in phospholipid bilayer membranes.     

Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens

The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an alpha-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide-polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide-peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.     

抗菌肽对种植体周围炎主要致病菌生长抑制作用的研究

目的研究抗菌肽KSL及其衍生物KSL—W对种植体周围炎主要致病菌的体外抑菌效果。方法应用二倍稀释法检测KSL和KSL—W对血链球菌、具梭核杆菌和牙龈卟啉单胞菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）;MTT法检测KSL和KSL—W对成骨样细胞MG-63的细胞毒性。结果KSL和KSL—W对具梭核杆菌的MIC和MBC分别为0．0156mg／mL和0．0313mg／mL,对牙龈卟啉单胞菌的MIC和MBC分别为0．125mg／mL和0．5mg／mL,在0．5mg／mL的浓度范围内对血链球菌没有抑制作用;KSL和KSL-W在0．5mg／mL的浓度范围内没有细胞毒性。结论KSL和KSL—W没有细胞毒性,对具梭核杆菌和牙龈卟啉单胞菌具有抑制作用。     

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.     

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.     

Interaction of antimicrobial peptides from Australian amphibians with lipid membranes

Solid-state NMR and CD spectroscopy were used to study the effect of antimicrobial peptides (aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1) from Australian tree frogs on phospholipid membranes. 31P NMR results revealed some effect on the phospholipid headgroups when the peptides interact with DMPC/DHPC (dimyristoylphosphatidylcholine/dihexanoylphosphatidylcholine) bicelles and aligned DMPC multilayers. 2H NMR showed a small effect of the peptides on the acyl chains of DMPC in bicelles or aligned multilayers, suggesting interaction with the membrane surface for the shorter peptides and partial insertion for the longer peptides. 15N NMR of selectively labelled peptides in aligned membranes and oriented CD spectra indicated an alpha-helical conformation with helix long axis approximately 50 degrees to the bilayer surface at high peptide concentrations. The peptides did not appear to insert deeply into PC membranes, which may explain why these positively charged peptides preferentially lyse bacterial rather than eucaryotic cells.     

富含色氨酸抗菌肽对种植体周围炎主要致病菌生长抑制作用的研究

目的研究富含色氨酸短肽Pac-525对种植体周围炎主要致病菌的体外抑菌效果。方法应用二倍稀释法检测Pac-525对血链球菌、具核梭杆菌和牙龈卟啉单胞菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)并绘制其Kill-time曲线。结果 Pac-525对血链球菌的MIC和MBC分别为0.125mg/mL和0.5mg/mL,对具核梭杆菌的MIC和MBC分别为0.007 8mg/mL和0.015 6mg/mL,对牙龈卟啉单胞菌的MIC和MBC分别为0.062 5mg/mL和0.125mg/mL;Kill-time曲线显示,Pac-525可以降低3种细菌的生长速度并呈现一定的量效关系,当浓度达到MBC时可完全抑制细菌在培养液中的生长繁殖。结论 Pac-525对种植体周围炎主要致病菌血链球菌、具核梭杆菌和牙龈卟啉单胞菌均具有抑制作用。     

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Natures antibiotics: the potential of antimicrobial peptides as new drugs

Animals and plants make a variety of substances to prevent potentially lethal infections. These include small antibiotic proteins, or peptides, which target bacteria, fungi and viruses. Research into these peptides not only give us an insight into how we naturally prevent infections, but can also provide us with new drugs to treat the ever-increasing danger of infections caused by antibiotic-resistant bacteria.     

Solid-state NMR study of antimicrobial peptides from Australian frogs in phospholipid membranes

Antimicrobial peptides, isolated from the dorsal glands of Australian tree frogs, possess a wide spectrum of biological activity and some are specific to certain pathogens. These peptides have the capability of disrupting bacterial membranes and lysing lipid bilayers. This study focused on the following amphibian peptides: (1) aurein 1.2, a 13-residue peptide; (2) citropin 1.1, with 16 residues; and (3) maculatin 1.1, with 21 residues. The antibiotic activity and structure of these peptides have been studied and compared and possible mechanisms by which the peptides lyse bacterial membrane cells have been proposed. The peptides adopt amphipathic -helical structures in the presence of lipid micelles and vesicles. Specifically ¹⁵N-labelled peptides were studied using solid-state NMR to determine their structure and orientation in model lipid bilayers. The effect of these peptides on phospholipid membranes was determined by ²H and ³¹P solid-state NMR techniques in order to understand the mechanisms by which they exert their biological effects that lead to the disruption of the bacterial cell membrane. Aurein 1.2 and citropin 1.1 are too short to span the membrane bilayer while the longer maculatin 1.1, which may be flexible due to the central proline, would be able to span the bilayer as a transmembrane -helix. All three peptides had a peripheral interaction with phosphatidylcholine bilayers and appear to be located in the aqueous region of the membrane bilayer. It is proposed that these antimicrobial peptides have a "detergent"-like mechanism of membrane lysis.This paper was submitted as a record of the 2002 Australian Biophysical Society     

Effect of antimicrobial peptides from Australian tree frogs on anionic phospholipid membranes

Skin secretions of numerous Australian tree frogs contain antimicrobial peptides that form part of the host defense mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al. (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as novel antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1, which exist as random coil in aqueous solution but have alpha-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC ( d 54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid headgroups by deuterium and (31)P NMR, respectively. In this report we demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterized using surface plasmon resonance (SPR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides has significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface interaction for aurein 1.2 and pore formation rather than membrane lysis by the longer peptides.     

Three new antimicrobial peptides from the scorpion Pandinus imperator

Three novel cysteine-free venom peptides, which were referred to as Pantinin-1, Pantinin-2 and Pantinin-3, respectively, have been identified from the scorpion Pandinus imperator by cDNA cloning strategy. The precursor of each peptide consists of a signal peptide, a mature peptide with no disulfide bridges, and an acidic propeptide with a typical processing signal. Each of the three peptides is an α-helical, cationic and amphipathic molecule with 13 or 14 amino acid residues. Their amino acid sequences are homologous to those of some 13-mer antimicrobial peptides isolated from scorpions. Antimicrobial assay showed that all the three peptides possess relatively strong activities against Gram-positive bacteria and a fungus, but have very weak antimicrobial activities against Gram-negative bacteria. Toxicity assay showed that the three peptides exhibit very low or mild hemolytic activities against human red blood cells. It is interesting to see that Pantinin-3 is able to potently inhibit the growth of vancomycin-resistant Enterococcus (VRE) S13, a pathogen that can cause a number of human infections; this suggests that Pantinin-3 has great potential to be applied in the treatment of VRE infections. Our findings gain new insights into the structure/function relationships of the small linear cationic antimicrobial peptides from scorpions, and provide new templates for designing of antimicrobial agents targeting antibiotic-resistant pathogenic bacteria.